Instead of tampering with the whole microstructure of titanium alloy base structure, direct laser metal deposition (DLMD) method can be used to target and solve a particular problem. This will result in extended application of titanium alloy. The enhancement of titanium alloy service life span can be done by fabricating composite coatings on titanium alloy. Additive manufacturing technique has been used over the years to repair and extend components’ life span. Experimental procedure was done at the National Laser Center, CSIR, South Africa with Ytterbium Laser System inbuilt with 3000 W for the quaternary coatings. Materials characterization was done according to the standard procedure. The stable beta phase in the copper reinforcement is very strong and this phase is propagated during the direct laser metal deposition process. The initiation and propagation of phases of beta-titanium structures (β-Ti) was due to the reaction in the molten pool of reinforcement copper and titanium alloy base. Also, aluminium-copper structures formed and brought up dendritic grain propagation as a result of feed rate of the reinforcement power, laser power and scanning velocity increase. The significant role played by the aluminium reinforcement in the lattice structures led to the titanium-aluminides (TiAl) propagation. The visibility of the dendritic phases as seen in the microstructures came about as aluminium reinforcement reacts with copper in the matrix as the molten pool solidifies. The properties were enhanced as a result of optimized process parameters. The manufacturing of Novel composite coatings is possible as indicated in this research. This novel composite coatings data is viable for applications in aerospace industry because of good metallurgical properties, enhanced mechanical and structural performance.•Aluminium-copper structures formed and brought up dendritic grain propagation as a result of feed rate of the reinforcement power, laser power and scanning velocity increase. Copper as a reinforcement had been reported to have strong stabilized beta (β) particles. Likewise, beta-titanium phase (β-Ti) propagated as a result of the fusion of titanium alloy and copper reinforcement. Moreover, intermetallic inter-dendritic eutectic phases network was formed by the combined beta and alpha phase of copper and aluminium reinforcements. These results would help materials engineer, Thermal engineers, and metallurgical engineers to understand the metallurgical evolutions of this quaternary composites.•When the process parameters are optimized, the quaternary composite coatings can be used by direct laser metal deposition technique to enhance the microstructures, mechanical properties and surface integrity of Titanium alloy for aerospace application. These data would help engineers in the aerospace industry to understand the influence of optimized process parameters on the metallurgical evolution.•One of the visible phases in the microstructure is Titanium-aluminide compounds which is responsible for the enhancement in hardness property. The composition of the novel composite coating also attributes to the enhancement of the surface integrity. Increase in Hardness property (528 HV) by 32.2% and 32.13% increase in the ultimate tensile strength (5179 MPa) came about as a result of the optimized process parameters at scan velocity of 1.2 m/min and laser power of 900 W. Silicon was at 13 wt% and Copper at 6 wt% (Ti-Al-Si-Cu) to achieve this enhancement. These data help metallurgical engineer to understand quaternary Ti-Al-Si-Cu composites for various application in aerospace industry.12. This is linked to the scan velocity and the laser power. The significant role played by the aluminium reinforcement in the lattice structures led to the titanium-aluminides (TiAl) propagation as shown in 23 using OMAX 5500 waterjet machine. The characterization of the materials was done in order to show the microstructural evolution and all standard procedures were observed during the characterization. The etching of the samples was made possible by Nital reagent. Washing of the samples with water was followed and the samples were air dried. Desiccator in the Laboratory was used to store the etched samples. Thereafter, experimental procedure then began. The experimental procedure used average particle size of 90 μm for the reinforcements . The mixture of the reinforcements was carried out at different weight percent via measuring scale (Shimadzu). Ti-Al-Si-Cu as follows: Ti-Al-11Si-5Cu , Ti-Al-11Si-5Cu , Ti-Al-9Si-3Cu , Ti-Al-9Si-3Cu , Ti-Al-7Si-1Cu , Ti-Al-7Si-1Cu , Ti-Al-12Si-2Cu , Ti-Al-12Si-2Cu , Ti-Al-13Si-6Cu , Ti-Al-13Si-6Cu , Ti-Al-7Si-4Cu , Ti-Al-7Si-4Cu . The powders were manufactured from TLS Company, Germany and the purity of the powders is 99.98%.The design of the experiment started by cutting Twelve (12) titanium alloy into dimension of 78 × 78 × 5 mmThe shape of the powders was manufactured as spherical. After the mixing of the reinforcements was done on the measuring scale, they were put inside a round container and paced inside a mixer (Tubular). The velocity of rotation of the mixer was set at 72 rpm and left rotating for 22 h. This was done to achieve homogenous mixture of the three reinforcements. The parameters for the experimental procedure were optimized via design of experiment. The following parameters were optimized: powder feed rate (2.5 g/min), gas shield rate (2.5 L/min), velocity of scanning (1.0–1.2 m/min), and laser power (900 – 1000 W) as shown in The authors followed universally expected standards for ethical behavior in conducting and publishing scientific research.Yonas Tibebu Mekonnen: Conceptualization, Methodology, Investigation, Formal analysis, Visualization, Validation, Writing – original draft. Endelkachew Addis Mekonen: Validation, Formal analysis, Visualization, Writing – review & editing. O. Fatoba: Conceptualization, Methodology, Investigation, Formal analysis, Visualization, Validation, Writing – original draft.The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.


The Copenhagen Burnout Inventory (CBI) has demonstrated good psychometric properties among different populations, but there is no known data on its validity among Russian-speaking medical students. The CBI-Student Survey focuses only on fatigue, but measures exhaustion in four different life domains: Personal Burnout (PB), Studies-Related Burnout (SRB), Colleague-Related Burnout (CRB), and Teacher-Related Burnout (TRB).To investigate the psychometric properties of the Russian version of the Copenhagen Burnout Inventory–Student Survey (R-CBI-S).A cross-sectional study was carried out among 771 medical students at Astana Medical University . Statistical analyses included test-retest reliability, internal consistency, item analysis, convergent and concurrent validity, and confirmatory factor analysis. Concurrent validity was evaluated by bivariate correlations of R-CBI-S with anxiety, depression, and satisfaction with the study.Test-retest reliability showed an ICC of 0.81. All item-total correlations for the total scale were positive (range 0.31–0.76). The Cronbach’s alpha coefficient was 0.94 . The Barlett’s sphericity test result was significant (p < 0.001), and the KMO measure of sampling adequacy exceeded 0.947. Convergent validity analysis results: PB , SRB , CRB , TRB . The R-CBI-S achieved good levels of goodness-of-fit indices .The test results indicated that the R-CBI-S scale appears to be a reliable and valid instrument. The R-CBI-S may be a useful tool in future research to identify burnout factors based on specific life domains for developing effective prevention measures among medical students. Burnout is a growing epidemic among medical students, which has been shown to have psychological and performance-related detriments . MedicalSeveral methods have been developed to study burnout among students, namely the Maslach Burnout Inventory–General Survey for Students MBI-SS; , the OldIn recent years, the CBI has been validated in different countries and study populations, such as university professors and academic staff members at Brazilian public universities , an acadThis study aims at evaluating the reliability and validity of the Russian version of the Copenhagen Burnout Inventory–Student Survey (R-CBI-S) in a sample of medical students at the Astana Medical University, Kazakhstan.(www.1ka.si) during the period October–December 2019. The questionnaire was completed by 771 students (response rate 40%). Of the participants, 25.0% were male. Academic year distribution among students was 1 year (218), 2 year (137), 3 year (125), 4 year (62), 5 year (60), and 6 year (169). The average age of the respondent was 20.7 years (ranged in age from 18 to 33).All medical students at any stage of their medical education at Astana Medical University were eligible to participate. Participants were invited via the “messenger” app and the university’s information portal, Sirius, to fill out an online questionnaire created on the 1ka platform The Copenhagen Burnout Inventory–Student Survey was converted into the Russian language from the original English version using a forward-backward translation process performed by specialists in the field of psychology and language. The final questionnaire was revised based on feedback from a sample of 20 participants through a pilot study.Data analysis was conducted using Microsoft Excel 2007, SPSS version 20.0, and Jamovi version 1.2.17. A statistically significant difference was accepted at a p-value of less than 5%.The reliability of the scale  was evaluated using the intraclass correlation coefficient (ICC). According to Internal consistency was evaluated by the total scale and subscales reliability analysis reflected by Cronbach’s alpha coefficient. A Cronbach’s alpha coefficient with a value of ≥ 0.7 is acceptable . CorrectConvergent validity was checked with average variance extracted (AVE) and composite reliability (CR). Values of 0.5 or more for AVE and 0.6 or more for CR were considered as having significant convergent validity . ConcurrConstruct validity was established by the confirmatory factor analysis (CFA) technique, with Bartlett’s test of sphericity and the Kaiser-Meyer-Olkin (KMO) measure of sampling adequacy used to test the dataset for factor analysis suitability. The CFA is used to assess the overall goodness of fit: the Root Mean Square of Error Approximation RMSEA (< 0.08); the Comparative Fit Index CFI(> 0.9); and the Tucker-Lewis Index TLI (> 0.9) .The Russian version of the Copenhagen Burnout Inventory was adapted for students. The R-CBI-S consists of 25 items that represent four dimensions: Personal Burnout (PB) — 6 items , Studies-Related Burnout (SRB) — 7 items , Colleague-Related Burnout (CRB) — 6 items , and Teacher-Related Burnout (TRB) — 6 items . The answers that can be given to each item are “always,” “frequently,” “sometimes,” “rarely,” and “never.” The scores attributed to these answers are 100, 75, 50, 25, and 0% respectively, with inverse scoring for item 10. For each scale, a total average score was calculated. According to Kristensen’s criteria of burnout levels, scores of 50 to 74 are considered moderate, 75–99 high, and a score of 100 is considered severe burnout .Table 1.The final translated Russian version of the R-CBI-S is presented in The test-retest reliability showed an ICC of 0.81 (CI 95% 0.63–0.94) for the R-CBI-S. The overall Cronbach’s alpha coefficient of the R-CBI-S was 0.939 , which indicates a high level of internal consistency. Corrected item-total correlation is shown in Figure 1 shows the factor model. Analysis of the eigenvalues indicated that four factors extracted with values above 1.0  according to The Barlett’s sphericity test result was significant (p < 0.001), and the KMO measure of sampling adequacy exceeded 0.947. Extracted AVE and CR from convergent validity analysis showed in Table 2 shows the correlations of the R-CBI-S with other variables. Weak positive correlations were found for R-CBI-S and GAD-7, PHQ-9. Satisfaction with the study was found to be negatively associated with R-CBI-S.The total R-CBI-S mean score was 39.96, and the mean subscale scores for this sample were 52.62 (PB), 50.93 (SRB), 23.50 (CRB), and 32.77 (TRB).Analysis of the literature showed that burnout is an important component of medical students’ mental health, which can affect the learning process and have further professional consequences. A feature of the CBI is that it divides burnout into four components. This makes it possible to identify predictors of burnout covering not only exhaustion, but dividing it into personal, studies-related, colleague-related, and teacher-related burnout, ultimately to draw up a more comprehensive approach to organization of the educational process. We agree with the opinion of The purpose of this study was to examine the psychometric properties of the Russian version of the CBI-S. Following adaptation and psychometric tests, this study found that the survey was reliable and valid for assessing burnout among Russian-speaking medical students in Kazakhstan. The ICC analysis showed that the R-CBI-S had high stability within 2 weeks of the test-retest (mean ICC 0.81).The internal consistencies of the four subscales were satisfactory, with all the Cronbach’s alpha values ranging from 0.874 to 0.926, and Cronbach’s alpha for R-CBI-S being 0.939. These results are slightly lower than those reported by AVE for all dimensions was equal to or more than 0.5, suggesting an adequate level of convergent validity. The CR values of the R-CBI-S constructs ranged between 0.86 and 0.88, which indicates a high level of convergent validity.The R-CBI-S was associated with anxiety, depression, and satisfaction with the study, lending support to the scale’s concurrent validity. A validated Chinese version of CBI was correlated not only with anxiety and depression, but also with physical distress and social support .The R-CBI-S demonstrated satisfactory construct validity, as tested by CFA. The results indicated that most fit indices were in acceptable ranges. Sufficiency of the model was demonstrated by Bartlett’s test of sphericity and the KMO measure.The R-CBI-S appears to be a reliable and valid instrument in measuring medical students’ burnout. The instrument could be useful for future efforts to develop an effective preventive intervention for burnout syndrome determination among Russian-speaking medical students.


The optical response of metal nanoparticles is governed by plasmonic resonances, which are dictated by the particle morphology. A thorough understanding of the link between morphology and optical response requires quantitatively measuring optical and structural properties of the same particle. Here we present such a study, correlating electron tomography and optical micro-spectroscopy. The optical measurements determine the scattering and absorption cross-section spectra in absolute units, and electron tomography determines the 3D morphology. Numerical simulations of the spectra for the individual particle geometry, and the specific optical set-up used, allow for a quantitative comparison including the cross-section magnitude. Silver nanoparticles produced by photochemically driven colloidal synthesis, including decahedra, tetrahedra and bi-tetrahedra are investigated. A mismatch of measured and simulated spectra is found in some cases when assuming pure silver particles, which is explained by the presence of a few atomic layers of tarnish on the surface, not evident in electron tomography. The presented method tightens the link between particle morphology and optical response, supporting the predictive design of plasmonic nanomaterials. A method linking three-dimensional morphology and quantitative optical scattering and absorption cross-sections of individual plasmonic nanoparticles is demonstrated, enabling a quantitative understanding of the morphology–optical response relation. This enables a wide tuneability using a single material, such as silver or gold, just by size and shape control,8 Numerical modelling of the optical response based on a 3D reconstruction from electron tomography was shown in ref. 10 Furthermore, gold–silver core–shell NPs were investigated, either showing simulations for a given morphology11 or comparing simulations with measured scattering spectra as function of shell thickness.12 However, the above works did not attempt an accurate comparison of measured and simulated cross sections, and focussed on the spectral features instead. Over the past years, we have developed a measurement and data analysis method to retrieve accurately quantitative cross-section spectra.13,14 In ref. Previous studies of correlative single-NP optical–electron microscopy using scattering spectra show the complex and sensitive dependence of the optical response on the morphology.16–18 including decahedra, tetrahedra and bi-tetrahedra. Similar to their gold counterparts,19,20 their response is ruled by localized surface plasmon resonances. The chemical reactivity of silver surfaces makes these systems attractive for catalysis applications,21,22 but also provides a route to chemical surface modifications which can be difficult to identify in TEM images while significantly modifying the optical response.23 We find here that an accurate quantitative study of cross-section spectra correlating experiment with simulation can uncover such detail. The presented case study on the one hand assesses the level of accuracy that can be achieved by our cross-section measurement method, and on the other hand exemplifies the kind of fine information that can be extracted from quantitative cross-section spectroscopy. Ultimately such progress might enable to reliably extract the 3D morphology of metal NPs from optical measurements alone.In the present work, we study faceted silver NPs produced by photochemically driven colloidal synthesis,2et al.17 and Pietrobon and Kitaev.16 As shown in 3 in aqueous solution are thought to aggregate to form decahedra under irradiation by a high power light-emitting diode (LED) centred at a 447 nm wavelength (violet spectrum in the graph). The formation of decahedra can be monitored via the progressive red shift of the extinction peak of the NP solution from spherical seeds (dashed line) to decahedra (solid line). Further details of the fabrication process and a kinetic study are reported in the ESI section S.I.†Let us present the workflow of the experiment summarized in 2 film (refractive index n = 1.46) supported by a 200 nm-thick Si3N4 film with 50 × 50 μm2 square windows, on a silicon substrate  triethoxysilane (APTES) (Sigma Aldrich) was centrifuged at 20k RCF for 20 min to spin down any large debris. 100 μL of this APTES stock was then diluted in 9.9 mL ethanol  to obtain a 1% APTES solution, in which the grid was incubated for 1 hour. The grid was then washed three times in ethanol followed by three times in water. The resulting functionalised grid was dried in air at 55 °C for 30 min and stored at 4 °C for no longer than one month. The decahedra solution  was wet-cast  composed of a 40 nm-thick SiOn = 1.52) between a microscope slide  and a coverslip  using a 0.5 mm thick adhesive silicone spacer (Grace Bio-Labs 664507), with the TEM grid surface facing the coverlip side. We chose anisole rather than microscope immersion oil as it is volatile and evaporates without leaving residuals, enabling subsequent electron microscopy. This assembly is mounted onto an inverted optical microscope  with a 40× dry objective  of 0.95 numerical aperture (NA) as depicted in To provide the NPs with a nearly homogeneous optical environment for the cross-section measurements, the TEM grid was sealed in anisole  equipped with a ruled plane diffraction grating  of 78 mm square size and 100 lines per mm. Spectra were acquired with a Peltier-cooled back-illuminated charge-coupled device (CCD) sensor . The spectrometer images the entrance slit onto the sensor, allowing to use the zeroth order of the grating to provide an image of the sample to select a specific particle for spectroscopy. The entrance slit acts as a spatial filter in the horizontal direction , whereas in the vertical direction the binning of the CCD sensor itself is used to define a region of interest. Together these define a 1.0 × 1.0 μm2 square region centred on the NP of interest from which the signal is collected. The corrections required to account for this finite region of detection are described in section S.III of the ESI.†The procedure for the optical measurements and the quantitative analysis of the optical cross sections is largely the same we adopted in ref. Within the transillumination scheme adopted, we define two imaging modalities based on the angular range of the illumination, as illustrated in σ = P/Iexc. Thus, a careful referencing to the exciting intensity24 of the single-particle extinction and scattering spectra enables us to measure accurately the magnitude of the cross sections. Note that the BF extinction signal includes contributions of both absorption and scattering, which have to be unravelled based on the scattering-only DF signal. Such retrieval procedure is presented in ref. η and ζ. η concerns the detection, and is the fraction of the total scattering collected by the objective. We note that η depends on the angular range of the illumination, such that ηBF≠ηDF; however, the difference is small for the decahedra, whose response is governed by the same dipolar mode under both BF and DF illumination. ζ concerns the excitation, and is the BF-to-DF ratio of the scattered power; it depends therefore on the relative intensity of the BF-to-DF illumination  and σabs(λ) refer to a given illumination and collection range. Specifically, in this work we measure σDFsca and σBFabs, which differ24 from the cross sections under plane-wave excitation.The optical cross sections are defined as the power removed from the exciting beam per excitation intensity: 25,26 The resulting reconstructions are down sampled by a factor 12 and segmented using the Otsu method to export them as .stl files, such as the one shown in As illustrated by n = 1.52). We neglected the small index mismatch between the thin silica window (n = 1.46) and anisole and used a homogeneous medium instead of a multi-layered structure, therefore the model used here is equivalent to the one described in the ESI of our previous work ref. d = 0 nm). This simplification allowed us to automate the importing and alignment of particle geometries from HAADF-STEM tomography into COMSOL. The stationary solution of Maxwell's equations under plane-wave (PW) excitation of given frequency, polarization, and propagation direction computed by COMSOL determines the spatial distribution of the electromagnetic field .The optical response of the particles is computed in the frequency domain using COMSOL Multiphysics®, a commercial software implementing the finite-element method. In the model, the NP is defined as silver using the permittivity reported in ref. σabs, σsca, η, ζ) from this solution.  where  is the conduction current in terms of the AC electrical conductivity ς. We integrate QJ over the NP volume to compute the absorbed power Pabs, and hence σPWabs = Pabs/Iexc dividing by the excitation intensity Iexc. The near-field solution can be projected to the far field via the far-field transform available in COMSOL, resulting in an angular distribution of the field  such as the one shown in Let us now discuss how we derive the observables of interest  – albeit not precisely along it, due to a tilt of a long axis of the particle, along which its polarizability is maximized. The far-field Poynting vector   and the collected fraction of scattering η.A dipole-like emission pattern is seen, with the dipole oriented close to the σ and η are computed under PW excitation, which we have indicated with the PW superscript; in the experiment instead we use the incoherent illumination produced by a high-NA condenser, which is composed of a wide range of directions. To reproduce the measured σDFsca and σBFabs we therefore perform and average a large number of PW simulations sampling the directional range of illumination (either BF or DF), each direction being assigned an appropriate weight according to the angular dependence of the illumination intensity in our microscope, that we have characterised. The unpolarised illumination is reproduced by averaging for each direction the results of two PW simulations with orthogonal excitation polarisation, namely p and s with respect to the imaged sample plane. An analogous directional averaging is applied to compute the scattering parameters  appropriate to the experimental angular ranges of illumination and detection. The mathematical details of such procedure are given in ref. We emphasize that these values of σDFsca and σBFabs spectra, which are shown in This averaging results in the 3V. †Twenty particles were measured in total, which we numbered with increasing volume 18,28 The quantitative comparison between measurements and simulations shows a remarkable agreement, considering that no adjustable parameters have been used. The difference between simulated and measured peak positions can be seen in the inset, and separately in (c). We find good correlation, with most particles showing a red shift of the measurement relative to the simulation by a few tens of nanometres. This finding is reminiscent of the shift observed in experiments with silver cubes.15 The relative deviation between simulated and measured peak magnitude (see panel d) shows a significant fluctuation, mostly with the simulation being higher, though the deviation decreases for large particles. Generally, the signal-to-noise ratio in the HAADF-STEM projection images is smaller for smaller particles, allowing for a larger relative error. In addition, the finite angular range used for the tomography reconstructions gives rise to a so-called missing wedge artefact, a result of a lack of information along certain directions. This can lead to systematic errors depending on the particle morphology, which could cause particle to particle fluctuations. On the optical measurement side, smaller cross sections are more affected by noise due to diffuse background scattering. However, the noise level is typically not significant in the present data, as can be seen in the scattering spectra shown in To correlate the results of experiment and simulations across all particles measured, we compare key spectral features in 29 silver cubes,15,30 and gold decahedra.31 We note that the samples were shipped from the optical experiment at Cardiff to the electron tomography at Antwerp in nitrogen atmosphere in a sealed container at room temperature, providing up to 4 days during which such rounding might have developed.32 In the area shaded in blue the simulated peak is also blue shifted, but the simulated amplitude is higher than the experimental one. Based on our previous work15 this is likely due to a surface layer forming on the particles. An increased damping in the permittivity can also lead to a decrease of the scattering cross section, as we shall discuss in subsection 3.2 below. The green area corresponds to a red shift of the simulated spectra with respect to the experimental ones, with an increase in amplitude. The two particles in this area show rather small deviations, within the accuracy of determining the values.In Below we investigate some of these potential sources of deviation in more detail on two selected particles. As the cross-section simulations taking into account the wide NA range of the microscope illumination are computationally expensive, we increased the sampling step size of the illumination direction from 0.21 NA to 0.3 NA, reducing the simulation time by a factor of two, while affecting the cross-section spectra by less than a few percent.3.1The measured NP morphology dictates the simulated optical cross sections, and thus should be as accurate as possible. In our analysis pipeline, the reconstruction of the electron tomography depends on analysis parameters which influence the resulting morphology. As mentioned earlier, electron tomography suffers from the missing wedge artefact, which leads to a lack of information along certain directions, and we found that the resulting morphology slightly depended on the number of iterations in the reconstruction process. One can also include pre-processing of the data such as smoothing procedures. In addition, to achieve a reasonable simulation time, the NP morphology needs to be meshed with an acceptable number of elements, which depends on the computational power available and the accuracy required. In this section we discuss the influence of these points on the reconstructed morphology and simulated spectra.N = 12. Downsampling by a factor N bins together pixels in an N × N × N volume, so reduces the number of elements defining the NP's surface by a factor of N2. The exact number of facets depended on the NP, but in general for R1 the NP's surface geometry consisted of a few thousand faces. In the reconstruction procedure R2, we smoothed the input projection images with a pixel radius of 3 prior to the iterations to improve the signal-to-noise ratio, and reduced N to 4, which increased the number of surface elements to tens of thousands. For reconstruction R3, we furthermore increased the iterations to 100 and reduced N to 1, which increased the number of surface elements to hundreds of thousands.We call R1 the meshed reconstructions used in versus the mesh size, as described in the ESI of ref. For the large number of surface elements resulting from R2 and R3, COMSOL was unable to reliably import the geometry and construct usable particle models. To circumvent this problem we reduced the number of surface elements to approximately 1000 before importing. This did not cause a significant loss of accuracy: we observed typically around 5 nm blue shift and 1% increase in amplitude  × 104 nm3 for , and the volume to surface ratios are V/S =  nm. For particle #3 the volumes are V =  × 104 nm3 and the volume to surface ratios are V/S =  nm.The reconstructions R2 and R3 resulted in a slightly altered geometry that was hardly discernable visually on the COMSOL mesh, so that we look here at the calculated volume and surface area changes  which in turn modify the measured cross sections slightly, as shown by the dashed lines.For the larger particle  on the Ag permittivity εexp(ω) measured by ellipsometry on a planar surface of polycrystalline Ag films27 as function of the angular frequency ω = 2πc/λ, with the speed of light c and the wavelength λ. We first fit εexp(ω) in the wavelength range between 400 nm and 700 nm, avoiding the Ag interband transitions at shorter wavelengths, with a Drude model, ε = ε∞ − ωp2/(ω2 + iωγ), as detailed in the ESI section S.VII.,ωp is the plasma frequency and γ is the damping. Then, we increase the damping by the term33–36gvF/R, where vF is the Fermi velocity, R is the effective radius, and g is a scaling factor. We use the radius R calculated from the particle volume V assuming a spherical shape, , resulting in R = 35.2 nm for particle #20 and R = 21.7 nm for particle #3. Finally, we add the permittivity change due to the increased damping to the measured permittivity data set, resulting in the modified permittivity εm(ω) = εexp(ω) + ε − ε to be used in the simulation.It is well known that the permittivity of a metal measured by ellipsometry on a planar surface can require a modification for NPs due to the reduced mean free path of the electrons.g from 0 to 1.5 , together with a broadening of the peaks, while the absorption cross section increases  are not significantly affected by the additional damping.The effect of the increased damping on the cross-section spectra is shown in 3.3σsca matching the measurements, we did not find a reconstruction that would consistently move the spectra of all particles enough that a further permittivity change could explain the remaining discrepancy. Therefore we consider here another deviation of the particle description in the model from reality, given by an atomically thin chemical surface modification, which is not expected to be visible in the electron tomography for the imaging settings used. Such a layer, which can form on silver (as opposed to gold) due to its reactivity, is most likely sulfide or oxide.37,38 Both compounds have a high refractive index and also absorption, causing a red shift and a decrease in scattering magnitude,15,39 mimicking the observed mismatch between simulation and measurement for the majority of the particles.While it might be possible that changing both reconstruction procedure and damping could produce a More discussion and data regarding the possible origin and experimental evidence and of such layers, including energy-dispersive X-ray spectroscopy, is given in the ESI section S.II.†Vc, with the remaining space in the original volume Vs providing the shell. The resulting average shell thickness h is taken as h = 2(Vs − Vc)/(As + Ac), where As and Ac are the surface areas before and after the scaling, respectively. Since sulfur is typically more reactive with Ag than oxygen, the wavelength-dependent permittivity of the shell was set to the one of silver sulfide.40To model such layers in COMSOL we used the following approach: starting from the surface mesh of the particle, we modeled a surface layer by isotropically scaling down the mesh, while fixing its center of mass, to define a Ag core of volume h = 1.0 nm. For particle #3 we used a scaling factor of 0.985, yielding h = 0.3 nm. These shell thicknesses yield a good agreement between simulated and measured scattering cross-section spectra as shown in e.g. #12, see ESI Fig. S10For particle #20 we scaled down the mesh by a factor of 0.97, creating a layer of thickness 415 to study individual silver nanohedra synthesized by photochemistry. Importantly, we extended the method to include electron tomography to determine the volumetric shape of the particles accurately, and used the resulting morphology and orientation for simulations of the quantitative optical cross-section spectra, for a fitting-parameter free quantitative comparison with the measured spectra. This is the first study of this type, combining fully quantitative optical cross-section measurements with correlative electron tomography determining quantitatively the 3D particle morphology and orientation, and corresponding quantitative simulations.We have used the pipeline for correlative and quantitative optical and structural electron microscopy characterization that we have recently developedWhile generally a good agreement of simulated and measured cross sections is found, quantitative differences are revealed. Specifically a red shift of the measurements compared to the simulations by a few percent, mostly for the larger particles, and a difference in magnitude, mostly a reduction for the smaller particles. To understand the origin of the deviations, the influence of three aspects was investigated. (i) The tomographic reconstruction method was examined, showing resulting morphology variations mostly for the smallest particles investigated. (ii) The addition of a realistic surface damping in the permittivity resulted in only slightly modified spectra. (iii) Adding a thin surface layer of tarnish, here modelled as silver sulfide, brought about, for realistic thicknesses in the 1 nm range, a match within the expected systematic errors. Let us emphasize that such conclusions would have been less stringent without the information on the cross-section magnitude. For instance, the red shift of the measured spectra can be explained both in terms of the geometry being more sharp, within the reconstruction accuracy, and by the tarnish layer; but only the latter hypothesis is in agreement with the measured cross-section magnitudes.The accuracy of the method can be improved going forward. For example, one could add polarization-dependent measurements and simulations, using linearly, radially, and azimuthally polarised light, where the latter has the advantage of only in-plane polarized excitation for both BF and DF, thus exciting the same resonances. Furthermore, the slight angular dependence of the objective transmission could be calibrated and taken into account. To avoid the formation of a tarnish layer, a similar study on gold nanohedra could be envisaged, allowing to isolate the accuracy of geometry and permittivity.This work and the adoption of the developed methodology paves the way towards an accurate quantitative understanding and verification of the morphology–optical response relation in plasmonic nanoparticles, especially for particles with complex shapes, which are important building blocks for next-generation devices.Y.W., P.B., and W.L. developed the workflow for correlative electron and optical microscopy. A.Z., P.B., and W.L. developed the quantitative optical microspectroscopy technique. Y.W. performed the sample preparation and optical microspectroscopy. A.Z., Z.S., P.B., and W.L. developed the numerical model and methods. Z.S. and A.Z. performed the numerical simulations. W.A. and S.B. performed electron tomography and analysis. A.Z., Z.S., and W.L. wrote the initial draft. All authors took part in interpreting the data and writing the manuscript.CRediT: Conceptualization W.L., P.B., W.A.; data curation Y.W., Z.S., A.Z., W.A., W.L.; formal analysis Y.W., Z.S., A.Z., W.A., W.L.; funding acquisition W.A., S.B., P.B., W.L.; investigation Y.W., Z.S., W.A.; methodology Y.W., Z.S., A.Z., W.A., P.B., W.L.; project administration W.A., S.B., P.B., W.L.; resources S.B., P.B., W.L.; software Z.S., A.Z., W.L.; supervision A.Z., S.B., P.B., W.L.; validation Y.W., Z.S., A.Z., W.A.; visualization Y.W., Z.S., A.Z., W.A., W.L.; writing – original draft Y.W., Z.S., A.Z., W.A., W.L.; writing – review & editing Y.W., Z.S., A.Z., W.A., S.B., P.B., W.L.https://doi.org/10.17035/d.2022.0176556287.Information about the data created during this research, including how to access it, is available on the Cardiff University data archive at The authors declare no conflicts of interest.NR-014-D2NR02131E-s001NR-014-D2NR02131E-s002NR-014-D2NR02131E-s003NR-014-D2NR02131E-s004NR-014-D2NR02131E-s005NR-014-D2NR02131E-s006NR-014-D2NR02131E-s007NR-014-D2NR02131E-s008NR-014-D2NR02131E-s009NR-014-D2NR02131E-s010NR-014-D2NR02131E-s011NR-014-D2NR02131E-s012NR-014-D2NR02131E-s013NR-014-D2NR02131E-s014NR-014-D2NR02131E-s015NR-014-D2NR02131E-s016NR-014-D2NR02131E-s017NR-014-D2NR02131E-s018NR-014-D2NR02131E-s019NR-014-D2NR02131E-s020NR-014-D2NR02131E-s021


Skin cutaneous melanoma (SKCM) is the world’s fourth deadliest cancer, and advanced SKCM leads to a poor prognosis. Novel biomarkers for SKCM diagnosis and prognosis are urgently needed. Long non-coding RNAs (lncRNAs) provide various biological functions and have been proved to play a significant role in tumor progression. Single-cell RNA sequencing (scRNA-seq) enables genome analysis at the single-cell level. This study explored prognostic lncRNAs in SKCM based on scRNA-seq and bulk RNA sequencing data.The TCGA cohort and melanoma samples in the GEO database  were included in this study. Marker genes were filtered, and ensemble lncRNAs were annotated. The clinical significance of selected lncRNAs was verified through TCGA and GEO dataset analysis. SiRNA transfection, wound−healing and transwell assays were performed to evaluate the effect of PRRT3-AS1 on cellular function. Immune infiltration of the selected lncRNAs was also exhibited.in vitro revealed that PRRT3-AS1 may be required for cancer cell migration in SKCM. PRRT3-AS1 was found to be related to epithelial-mesenchymal transition (EMT) signaling pathways. DNA methylation of PRRT3-AS1 was negatively related to PRRT3-AS1 expression and showed significant prognosis value. In addition, PRRT3-AS1 may suppress immune infiltration and be involved in immunotherapy resistance.A 5-marker-lncRNAs model of significant prognostic value was constructed based on GSE72056 and the TCGA cohort. PRRT3-AS1 combined with DANCR was then found to provide significant prognostic value in SKCM. PRRT3-AS1 was filtered for its higher expression in more advanced melanoma and significant prognosis value. Cellular function experiments PRRT3-AS1 may be a diagnostic and prognostic biomarker of SKCM. Skin cutaneous melanoma (SKCM) is one of the most fatal human diseases and the fourth leading cause of cancer-related mortality worldwide . SKCM raRNAs can be divided into protein-coding messenger RNAs and functional noncoding RNAs, the latter of which was once considered to be coded by “junk” DNAs. Recently, various functional noncoding RNAs, such as small nuclear RNAs and longer noncoding transcripts, have been discovered and described . Long noScRNA-seq enables genome-wide gene expression analysis with single-cell resolution, providing unprecedented capabilities in the identification of cellular heterogeneity, the transition of cellular states, and intercellular communications in complex tissue . Therefohttp://www.ncbi.nih.gov/geo) were downloaded as MINiML files. The extracted data were normalized by log2 transformation by the normalize quantiles function of the preprocessCore package in R software. Probes were converted to gene symbols according to the annotation information of the normalized data in correspond platforms. Marker genes were filtered from GSE72056, and ensemble lncRNAs were annotated. Selected lncRNAs were verified through TCGA and GEO dataset analysis. RNA-sequencing expression (level 3) profiles and corresponding clinical information were downloaded from the TCGA dataset. Raw data of selected GEO databases (http://www.ncbi.nih.gov/geo) were downloaded as MINiML files. The extracted data were normalized by log2 transformation by the normalize quantiles function of the preprocessCore package in R software. Probes were converted to gene symbols according to the annotation information of the normalized data in correspond platforms. Key functions of the selected lncRNAs were validated through a series of assays against cell model in vitro. All the R packages in the study were implemented by R software version 4.0.3. Original data including metadata of datasets involved, main R code and survival analysis results were collated as supplementary document   , 14. Tumhttps://hgserver1.amc.nl/cgi-bin/r2/main.cgi) based on the GSE19234 dataset. Genomics Analysis and Visualization Platform is an online datamining and discovery platform designed to perform datascience tasks in the omics field.Multivariate and univariate Cox proportional hazard regression of risk factors were performed to identify the proper terms to build the nomogram. The forest was used to show the Pvalue, HR and 95% CI of each variable through ‘forestplot’ R package. A nomogram was developed based on the results of multivariate cox proportional hazards analysis to predict the 1, 5, and 10-year overall recurrence based on the TCGA-SKCM cohort. The nomogram provided a graphical representation of the factors associated with the prognostic model through ‘rms’ R package. To further verify the predictive performance of the two most promising lncRNAs, we also performed survival analysis in the Genomics Analysis and Visualization Platform (http://gepia2.cancer-pku.cn/#index) (https://www.ncbi.nlm.nih.gov/gds). Copy number variation analysis of PRRT3-AS1 was conducted using the cBioPortal online tool   online tool (/#index)  was utilal.org/) . The cBi2 humidified incubator. Cells were plated into a 6-well plate at a density of 70–90%. The PRRT3-AS1 si-RNA (si- PRRT3-AS1) and its corresponding scrambled siRNA control (si-NC) were obtained from Miao Ling (China) (Two melanoma cell lines (A2058 and SK-MEL-28) were obtained from ATCC (American Type Culture Collection) and cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum  at 37°C in a 5% CO5 cells per well in 2 mL medium. After being transfected with siRNA, a straight linear wound was made in each well by using a 1 mL pipette tip . Then, the cells were carefully washed with PBS to remove cell debris and cultured in DMEM supplemented with 5% FBS. Finally, wound healing images were taken at 0, 24 and 36 h by using an inverted microscope  with a 200x objective.A wound healing assay was performed to evaluate cell migration ability. Cells were seeded overnight in 6-well plates at a density of 105 cells/well were inoculated above the 8 μm chamber  with a medium containing 200 μL serum-free medium and below the chamber with a medium containing 10% FBS of 500 μL for 24 h. Finally, five fields were randomly selected to capture images, and the number of cells passing through the chamber was counted.The migration capacities of cells were determined by a transwell assay. A2058 and SK-MEL-28 cells at a density of 1 × 10http://biocc.hrbmu.edu.cn/CancerSEA/home.jsp) (The Cancer Single-cells State Atlas (CancerSEA) online database (ome.jsp)  was accehttp://www.csbio.sjtu.edu.cn/bioinf/lncLocator/) (http://lncatlas.crg.eu/) (http://www.bio-bigdata.net/LnCeVar/search_quick.jsp) (PRRT3-AS1 location was explored using the lncLocator online tool (ocator/)  and the crg.eu/) . The lncick.jsp)  based onhttps://genome.ucsc.edu/), and the relationship between the expression of PRRT3-AS1 and the DNA methylation of PRRT3-AS1 was determined using the Xena online tool (https://xena.ucsc.edu/) (via the Scatterstats application in the Hiplot online tool (https://hiplot.com.cn/basic/ggscatterstats). OS was compared between the high-methylation group and the low-methylation group.In the TCGA-SKCM cohort, the chromosome location of PRRT3-AS1 was revealed using the UCSC Genome Browser online tool (sc.edu/) . UCSC coP < 0.05).Immune infiltration of PRRT3-AS1 in the TCGA-SKCM cohort was assessed by utilizing immunedeconv package. The ‘ggstatsplot’ package was used to draw the correlations between gene expression and immune score, the ‘pheatmap’ package was used to draw multi-gene correlation. RNA-sequencing expression (level 3) profiles and corresponding clinical information for PRRT3-AS1 were downloaded from the TCGA dataset. Potential ICB response was predicted with TIDE algorithm. TIDE uses a set of gene expression markers to evaluate two different mechanisms of tumor immune escape, including dysfunction of tumor-infiltrating cytotoxic T lymphocytes (CTLS) and rejection of CTLS by immunosuppressors. Patients with High TIDE score showed poor efficacy of immune checkpoint blockade therapy (ICB) and short survival after ICB . Used SpThe overall design and flowchart of this study are shown in P < 0.001). The median OS time of the high-risk group was 4.7 years, and that of the low-risk group was 8.9 years , DANCR , and age  were identified as significant positive prognostic factors for pT-stage  and pN-stage  , DANCR , and age  were identified as significant positive prognostic factors for pT-stage , pN-stage , and pM-stage  as well as significant positive prognostic factors for OS . The median DFS time of the high-risk group was 5.5 years, and that of the low-risk group was 10.4 years. The ROC curve also showed that the DFS of the high-risk group was lower than that of the low-risk group  and a low-risk group (n = 225). The disease-free survival (DFS) of the high-risk group was significantly lower than that of the low-risk group (P = 0.026)  and months of disease-specific survival analysis (P = 3.571e-4) were conducted, which indicated significantly poor prognosis associated with PRRT3-AS1 alteration in the TCGA-SKCM cohort , SNAI1 (P = 0.0055), and TWIST1 (P = 0.02) in the TCGA-SKCM cohort (PRRT3-AS1 expression was significantly positively correlated with EMT (r = 0.40) and invasion (r = 0.34) signaling pathways in the GSE81383 dataset . FurtherThe lncLocator illustrated that the cytoplasm contains most PRRT3-AS1 , T cell CD8+ , neutrophil , macrophage , and myeloid dendritic cell  were significantly enriched in the low-expressed PRRT3-AS1 group    silencing and methylation state are critical to the function of lncRNAs , 33. TheHowever, there were two limitations in our study. First, the SKCM data in this paper were from European and American populations, and it remains unclear whether the conclusions apply to other populations, especially Asian populations. Second, the underlying molecular mechanism of PRRT3-AS1 still needs to be further explored.In this study, we discovered PRRT3-AS1 is a diagnostic, prognostic, and immunotherapy biomarker of SKCM. PRRT3-AS1 plays an important role in SKCM promotion, and its potential as a therapeutic target for SKCM warrants further study.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.ST and WZ designed the study. ST reviewed the manuscript. WZ, XX, ZH, and XZ performed cell experiments and bioinformatic analysis. WZ wrote the manuscript. YL, K-LC, JZ, and ST are the guarantors of this work and, as such, have full access to all data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors approved the manuscript.This study was funded by the National Natural Science Foundation of China , Guangdong Medical Research Foundation Project , Shantou Science and Technology Project ([2019]10602), 2020 Li Ka Shing Foundation Cross-Disciplinary Research Grant , Guangdong University Innovation Team Project(2021KCXTD047), and Guangdong Science and Technology Special Fund .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Transient ischemic attack (TIA) provides a unique opportunity to optimize secondary preventive treatments to avoid subsequent ischemic stroke (SIS). Although atrial fibrillation (AF) is the leading cause of cardioembolism in IS and anticoagulation prevents stroke recurrence (SR), limited data exists about the risk of new-diagnosed AF (NDAF) after TIA and the consequences of the diagnostic delay. The aim of our study was to determine this risk in a cohort of TIA patients with long-term follow-up.We carried out a prospective cohort study of 723 consecutive TIA patients from January 2006 to June 2010. Median follow-up was 6.5 (5.0–9.6) years. In a subgroup of 204 (28.2%) consecutive patients, a panel of biomarkers was assessed during the first 24 h of the onset of symptoms. Multivariate analyses were performed to find out the associated factors of NDAF. Kaplan-Meier analysis was also performed to analyzed risk of SIS.P < 0.001) was associated with significant risk of NDAF during the first 5 years of follow-up. Patients with NDAF after admission and before 5 years of follow-up had the highest risk of SIS (P = 0.002).NDAF was indentified in 116 (16.0%) patients: 42 (36.2%) during admission, 18 (15.5%) within first year, 29 (25%) between one and five years and 27 (23.3%) beyond 5 years. NDAF was associated with sex  [hazard ratio (HR) 1.61 ], age [[HR 1.05 ], previous ischemic heart disease (IHD)  and cortical DWI pattern [HR 2.81 ]. In the Kaplan-Meier analysis, NT-proBNP ≥ 218.2 pg/ml (log-rank test The risk of NDAF after TIA is clinically relevant. We identified clinical and neuroimaging factors of NDAF. In addition, NT-proBNP was related to NDAF. Our results can be used to evaluate the benefit of long-term cardiac monitoring in selected patients. Transient ischemic attack (TIA) provides a unique opportunity to optimize secondary preventive treatments and avoid subsequent ischemic stroke (IS) . Up to 2The aim of our study was to determine the risk of NDAF after TIA in a cohort of consecutive TIA patients with long-term follow-up. We describe clinical characteristics, neuroimaging features and blood-biomarker patterns related to AF occurrence in order to identify those patients who would benefit from long-term cardiac monitoring. We also include a study of the prognostic repercussions of the diagnostic delay.2DS2-VASc risk score were recorded (The REGITELL registry (Registro de pacientes con ictus transitorio de Lleida [in Spanish]) methodology has been described in detail previously , 18. Conrecorded . Any potrecorded . All casrecorded . Patientrecorded , 28.Written informed consent or assent from relatives was obtained for all the participants. The study was approved by our local Ethics Committee: the “Comité d'Etica i Investigació Clínica de l'Hospital Universitari Arnau de Vilanova de Lleida” .AF was defined as a period of >30 s duration of an absolute arrhythmia without detectable P-wave activity and irregular RR interval . The wayStructured clinical visits were performed by a stroke physician  during the follow-up period to determine the risk of further vascular events. SR was defined by the appearance of new focal symptoms or signs associated with acute ischemic changes shown in neuroimaging scans (CT or MRI) made at 7 days, 3 months,1, 5, and 10 years. If a patient moved out of the local area or if travel to the hospital was impossible, the follow-up was conducted by phone. Recurrent events and NDAF were also actively identified by an annual review of electronic medical records .From January 2008 to June 2010, blood samples were obtained by standard venipuncture within the first 24 h after the onset of symptoms at the emergency department admission to test a panel of biomarkers  that inc2 score25, CHA2DS2-VASc10, outcomes and biomarker levels between non-AF, previous AF and NDAF patient groups. The quantitative variables were compared using either the student's T-test or the Mann-Whitney U test. The qualitative variables were compared using the chi-squared test or Fisher's exact test when the expected cell frequency was <5. Biomarkers were not normally distributed (P-P plot), and values were expressed as median (interquartile range). A Bonferroni correction (a multiple-comparison correction) was applied to all the significant associations to reduce the risk of finding false-positive associations. To calculate the sensitivity and specificity for biomarker cut-off values which allowed to discriminate TIA patients with NDAF from TIA patients without AF, a receiver operator characteristic (ROC) analysis was performed. The cumulative risks of NDAF after excluding patients with previous AF during follow-up were estimated using a Kaplan-Meier analysis. The results were censored at the time of the outcome event, patient death, or the end of the follow-up period. Risks were compared using the log-rank test. Data on patients with no information at 10 years were censored at the time of the last available follow-up. A Cox proportional hazards multivariable analysis was performed including clinical variables (model 1), clinical variables and neuroimaging features (model 2), clinical variables and biomarker data (model 3), and clinical variables, neuroimaging features and biomarker data (model 4) to identify predictors of new diagnosis of AF after TIA. It was also adjusted for patient characteristics that significantly predicted outcomes in univariate logistic regression models. We compared the risk of stroke recurrence in subgroups of patients categorized according to the main identified predictors using a Kaplan-Meier analysis and the log-rank test. All the tests were 2-sided. Missing data was included as a random effect when fitting the model for multivariable analyses. The statistical analysis of the data was carried out using the SPSS statistical package, version 24.0.  and Graphpad software version 6 (LLC).We compared the baseline characteristics, ASCOD classification, presence and distribution of acute lesions in DWI, ABCDA total of 723 patients were included in the analysis after excluding 48 patients who were diagnosed as mimics . Basic dp < 0.001], had a higher proportion of carotid territory events , speech impairment  and previous ischemic heart disease , but a lower proportion of non-definitive TIA events  and isolated sensory impairment  than patients without AF (p = 0.001), the CHA2DS2-VASc score  was higher than non-AF groups.Patients with NDAF were older  than non AF patients.New diagnosed AF patients had higher levels of NT-proBNP , age , previous ischemic heart disease , and cortical DWI lesion pattern    . A cut-oP = 0.002), patients with previous ischemic heart disease (IHD) (log-rank test P =< 0.001), patients with carotid territory events (log-rank test P = 0.003), and patients ≥ 75 years of age (log-rank test P < 0.001) had a significantly higher risk of NDAF throughout follow-up. In contrast, cortical DWI lesion patterns (log-rank test P < 0.001) and NT-proBNP ≥ 218.2 pg/ml (log-rank test P < 0.001) were associated with a significant risk of NDAF during the early follow-up, especially during the first 3–5 years.In the Kaplan-Meier analysis , female P = 0.095], of previous heart failure  and of positive DWI   vs. 22 (37.3%); P = 0.040]. In contrast, cortical lesion in one vascular territory was present in a greater proportion in NDAF patients  . HyperteWhen we analyzed the different categories of NDAF with respect to time of diagnosis from the onset of symptoms , we idenP = 0.002)  .In our study of consecutive TIA patients attended at an emergency department, we observed a non-negligible risk of new diagnosis of AF during the long follow-up. Nearly one out of five patients without previous diagnosis of AF developed a new-diagnosed AF. TIA provides an opportunity to change the natural evolution of cerebrovascular disease. The early diagnosis of AF facilitates the start of effective treatments for secundaru prevention such as anticoagulation . Howeverregistry.org project that reached 5 years of follow-up. Although AF-related brain ischemic events have been associated with disability strokes  and the Instituto de Salud Carlos III,  and the INVICTUS plus Research Network.This study was supported by the Catalan Autonomous Government's The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The quality of life at two years was significantly improved in the PPS when compared to the control group . No late adverse event was observed after cessation of the treatment. In this study, sixteen weeks of PPS monotherapy in women with recurrent UTI significantly reduced the numbers of recurrent UTI episodes during the 2-year follow-up.For women with recurrent urinary tract infection (UTI), previous U101 study has shown that pentosan polysulfate sodium (PPS) monotherapy for 16 weeks significantly reduced UTI episodes in the treatment group throughout the trial period. In this follow-up study, we aimed to assess whether the effects of PPS would last after completion of the trial to prevent recurrent UTIs. Conducted from 2018 to 2019, the U101 study was a multicenter, prospective, phase 2a, randomized trial, enrolling women with recurrent UTI to study the effects of a 16-week oral PPS monotherapy. After approximately two years, the follow-up was conducted by phone interview, obtaining data including self-reported UTI events, quality of life questionnaire, and adverse events. The primary endpoint of follow-up study was UTI recurrence-free survival and the secondary endpoints were quality of life and adverse events. Approximately two years after completion of the trial, the rate of recurrent UTI was 25% (3 of the 12 patients) in the PPS group and 85.7% (12 of the 14 patients) in the control group. Over the entire follow-up period, the UTI recurrence-free survival was significantly better in the PPS group than in the control group (log-rank test  Women with recurrent UTI, defined as at least two episodes of UTI in six months or three episodes in twelve months, are frequently exposed to antibiotics which might increase the risk of antimicrobial resistance and cause side effects from antibiotics6. Several guidelines and reviews have provided prevention strategies based on the best evidence for general or specific populations of women with recurrent UTI8. However, antibiotic prophylaxis for recurrent UTIs remains the most effective method, outweighing other nonantibiotic alternatives with unsatisfying effectiveness, such as daily cranberry pills, daily estrogen therapy, and acupuncture10.The lifetime risk of a woman developing a UTI is around 30–50%11. In the control arm, the median time to first confirmed recurrent UTI was 85.5 days and the overall UTI recurrence rate was 64% at the end of the trial. The high protection rate of PPS in the treatment arm led to early termination of patient enrollment of the trial and quick launching of a phase 3 randomized, double-blind, placebo-controlled study. Notwithstanding the observed in-trial benefits of PPS monotherapy, an extended follow-up study of the previous phase 2a U101 trial was planned to investigate the long-term protective effects of the treatment and the quality of life (QoL) of participants.Previously, we had published the outcome of a new nonantibiotic management for recurrent UTI in a multi-center Phase 2a, randomized trial using PPS as monotherapy, showing great efficacy of PPS in preventing recurrent UTI during the 16-week trial period11. The protocol was approved by the institutional review boards at National Taiwan University Hospital (IRB# 201802019MSC), Taipei Tzu Chi Hospital (IRB# 07-FS01-013), and Tri-Service General Hospital (IRB# 1-106-01-035) and conducted in accordance with the principles of Good Clinical Practices and the ethical principles of the Declaration of Helsinki. Written informed consent was obtained from patients before inclusion. The intervention drugs of oral PPS (Urosan®) and laboratory tests were sponsored by TCM Biotech International Corporation .The details of U101 study, including patients, methods, design, and execution have previously been publishedIn this multicenter, open-label, phase 2, randomized controlled trial, women with recurrent UTI were treated with oral PPS or observation. Between June 2018 to May 2019, a total of 27 women meeting the inclusion criteria who gave written informed consent were enrolled and underwent randomization  for 8 weeks and then with a maintenance dose of 100 mg twice daily (200 mg/day) for the following 8 weeks. In the post-trial period, PPS monotherapy was discontinued without any following prophylaxis for UTI in all patients. Treatment and follow-up for patients with recurrent UTI were under a consultant‐led appointment system with a patient-initiated system of care.12, along with at least two lower urinary tract symptoms  or at least one specific result  from urine analysis13.During the trial period, a diagnosis of UTI was made by a positive urine culture The post-trial data collection was conducted approximately two years after randomization for each participant through phone interviews amid the pandemic of severe acute respiratory syndrome coronavirus 2 diseases. For safety assessment, patients were actively asked to report any adverse events that occurred after completion of PPS treatment during the post-trial period. Severe adverse events would be recorded as any life-threatening event, in-patient admission to hospital, or important medical event. For monitoring long-term protective effects, the patients who remained free from recurrent UTI at the end of the trial in both groups were asked to report any recurrent UTI episodes. For patients with UTI recurrence, the date of the first UTI episode, the types of lower urinary tract symptoms, and the number of overall recurrent UTI episodes during the post-trial period would be recorded.15. Each dimension has three levels. Subjects were given three points for having no problems, two points for some problems, and one point for those who felt extremely problematic.The general QoL reflecting overall health status was assessed before trial entry and at post-trial follow-up survey. The score was subjectively selected by participants based on a 5-point scale, with 100 representing the participant's best imaginable health state and 0 representing the worst imaginable health state. A questionnaire of EQ-5D describing 5-dimensional health status was assessed along with QoL, including mobility, self-care, usual activities, pain/discomfort, and anxiety/depressionThe primary endpoint was UTI recurrence-free survival, defined as the time from randomization to a documented UTI event or censoring at the date of the last follow-up. In the post-trial period, the diagnosis of UTI was made by at least two self-reported lower urinary tract symptoms by participants clinically. Microbiological confirmation for recurrent UTI was not mandatory. The secondary analyses assessed the satisfaction and well-being of participants before and approximately two years after the trial. Secondary endpoints included self-reported adverse events and QoL assessment by QoL scores and EQ-5D scores.t-test. The difference between groups was done with Mann–Whitney U test and Student’s t-test for comparison of medians and means, respectively. All tests were 2-tailed and a p value of < 0.05 was considered significant. Statistical analyses were performed using the software: SPSS  and PRISM program .Endpoints analyses were done in the intention-to-treat (ITT) population. Follow-up was calculated by the standard Kaplan–Meier method for the construction of UTI-free curves. A log-rank test was used for the comparison of recurrence-free survival duration between groups. The difference in QoL and EQ-5D changes between 0 and 24 months were tested for normality with Kolmogorov–Smirnov test and skewness with Kurtosis, and statistical inference with Student’s two-sample The IRB of National Taiwan University Hospital (IRB# 201802019MSC), Taipei Tzu Chi Hospital (IRB# 07-FS01-013), and Tri-Service General Hospital (IRB# 1-106-01-035) all approved this study. All the procedures involving human participants followed in the study were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration.11. One patient in the PPS group withdrew consent and resulted in 26 patients included in the ITT analysis and follow-up investigation for primary and secondary outcomes at approximately two years after randomization. The overall median follow-up duration was 25.8 months (IQR 24.8–27.6). Baseline characteristics including age, frequency of UTI before screening, renal function, and underlying medical conditions were all similar between the control group and the PPS group  or the PPS group (n = 13) for the original U101 study . The hazard ratio for UTI recurrence-free survival was 0.11  for the comparison of the PPS group with the control group.The median duration of follow-up for recurrent UTI in the PPS group was 24.9 months (IQR 22.9–25.8). Only three out of twelve participants (25%) had self-reported UTI recurrence within 24 months during the follow-up period Table . In contAside from one serious adverse event of temporary hearing loss which resolved completely after steroid treatment during the trial period, no other drug-related adverse event was reported during the 2-year follow-up period.p = 0.16) and without skewness (Z = − 1.02). The mean QoL score at 24 months had increased by 15.0 points (p = 0.006) compared to 0 month before treatment in the PPS group (Table p = 0.045). For comparison between the two groups, the QoL score was significantly higher in the PPS group than the control group at 24 months (p < 0.001) but not at 0 month (p = 0.254)  at baseline 0-month (Table p = 0.001). EQ-5D score showed an improvement in the PPS group of 0.6 points  .The ITT analysis for EQ-5D-3L score did not show a significant difference between groups p = 0.01 at basp = 0.001) and anxiety/depression (p = 0.004) scores. No other subscores for each domain between two groups reached statistical significance at baseline 0 month or 24 months. The changes in EQ-5D-3L didn’t show a significant difference between the two groups.Scores in each dimension of EQ-5D among PPS and control group at 0 month to 24 months were shown in Fig. The follow-up results of the multicenter, open-label, prospective, phase 2a, randomized controlled trial demonstrated that 16-week PPS monotherapy significantly improved UTI recurrence-free survival for 2 years compared with the control arm. In this report, the recurrent rate in one year after the study was 16.7% (2/12 participants) in the PPS group and 64.3% (9/14 participants) in the control group. To our knowledge, this was the first clinical trial using PPS monotherapy for recurrent UTI prevention which showed promising results in both trial period and post-trial follow-up period.10. Antibiotic prophylaxis for 6 to 12 months reduced the risk of recurrence in women with recurrent UTI (at least 2 episodes in 12 months) to 12.3% compared with the recurrence rate of 65.5% in the placebo group16. Comparatively, antibiotics represented higher severe side effects with a relative risk of 1.58 (95% CI 0.47–5.28) than the placebo group. Besides, antibiotic prophylaxis loses the power of protection soon after discontinuation of the treatment16. In the 6 months follow-up period after an antimicrobial prophylaxis study, the recurrence rate in the antibiotic group and placebo-controlled group became identical17.Continuous antibiotic prophylaxis was the most efficacious method in preventing recurrent UTIs in the literature7. The possible mechanism is the proanthocyanidins in cranberries which can inhibit the adhesion of P-fimbriated E. coli to the urothelial cell receptor. Cranberry oral juice or tablet supplements might be offered according to availability and patients’ preference. A trial comparing two treatments, cranberry and lactobacillus, against the placebo group showed that at six months, 16% of women in the cranberry group, 39% in the lactobacillus group, and 36% in the control group had UTI recurrence for at least one episode18. However, in a comparative trial for five prevention strategies targeting patients with eight or more UTIs a year, an average of 4.4 recurrent episodes in a year occurred in the cranberry prophylaxis group, which was higher than 3.1 episodes in the daily estrogen group, 2.8 episodes per year in the acupuncture group, and 1.3 episodes in the antimicrobial prophylaxis group9. In another trial of 319 college women, the active cranberry prophylaxis for six months presented a higher recurrence rate of UTI by 20% versus 14% in the control group10. Nevertheless, cranberry prophylaxis is still recommended because of the safety profile of the food-based supplement.AUA/CUA/SUFU guidelines indicate cranberry prophylaxis for recurrent UTIs as a conditional recommendation with the evidence level Grade C19. However, several other agents for UTI prevention revealed controversial results of their efficacy7. The panel of AUA/CUA/SUFU guidelines could not find sufficient evidence to recommend other prevention methods such as lactobacillus, D-mannose, methenamine, herbal supplements, intravesical instillation of hyaluronic acid or chondroitin, and immunoactive prophylaxis7.Vaginal hormonal replacement is unanimously recommended by several clinical practice guidelines for peri- and post-menopausal women with recurrent UTIs20. In a randomized non-inferiority trial for UTI prevention, methenamine hippurate was as effective as low-dose antibiotics for women with a history of recurrent UTI21. However, nearly 56% of patients using methenamine hippurate still received therapeutic antibiotics for UTI during the 12-month treatment period. During the 6-month post-trial follow-up period, UTI rates and duration of antibiotic use were found to be higher in the methenamine hippurate group than in the low-dose antibiotic prophylaxis group.Methylamine salts appeared to be safe and effective as an alternative to antibiotics for the prevention of UTI in the literature24. The treatment aims to restore the important lining of urothelial epithelium which plays an important role in stopping bacterial invasion and adherence26. With the same concept, heparin intravesical instillation was studied for treating interstitial cystitis27. GAG replacement therapy demonstrated a durable protection effect for free from recurrent UTI for up to 47 weeks after 9 times of HA instillations over 6 months22. Other research revealed a less satisfactory result that only 48% of patients were free from UTI in 12 months after a combination intravesical instillation of HA and chondroitin sulfate23. Because of the high cost of HA and chondroitin sulfate and the requirement of catheterization for each bladder instillation, the treatment is unattainable in real-world practice.Glycosaminoglycan (GAG) replacement therapy, hyaluronic acid (HA) with or without chondroitin sulfate intravesical instillation, is thought to be promising in treating recurrent UTI in previous studies28 and binding to urothelium with sufficient strength against bladder washing29 in animal models. For patients with painful bladder syndrome/interstitial cystitis, the use of oral PPS was able to improve symptoms and repair abnormal bladder surface glycosaminoglycans30. Therefore, the oral administration of PPS brought great convenience to both clinicians and patients considering GAG replacement therapy with high compliance and long-term protective effect.PPS, a sulfated polysaccharide, was shown to be effective at restoring GAG-deficient bladder mucosa5. Women reported fear of unnecessary antibiotics and the development of antimicrobial resistance as well. Patients even reported frustrations with the limited effect of nonantibiotic prophylaxis strategies for recurrent UTIs. However, effective alternative prophylaxis, using oral immunostimulation with bacterial extracts of E. coli, could significantly improve the QoL of patients, including the reduction in anxiety and depression31. In this 2-year follow-up study, recurrent UTIs had an unfavorable effect on general QoL whereas QoL improvement was observed at 24 months after PPS treatment. In terms of EQ-5D scores, patients without PPS prophylaxis showed significantly lower scores than the PPS group in two dimensions, representing physical pain/discomfort and mental anxiety/depression. Other dimensions, including mobility, self-care, and usual activities, were not influenced by UTI before trial entry or at the 24-month follow-up.A qualitative analysis revealed that the patient attitudes toward antibiotic management of recurrent UTIs are dominated by fear and frustrationThe limitations of this phase 2a follow-up study were the small number of cases and lack of a placebo control arm. Therefore, a phase 3 double-blind, randomized, placebo-controlled trial has been launched to verify the results of this phase 2a study. During the follow-up period, the diagnostic criteria for UTIs were loose, without confirmation by positive urine culture, which might overestimate the number of UTI events. Nevertheless, the results still highlighted the fact that PPS monotherapy has a long-term effect in preventing recurrent UTI.The follow-up study showed that a 16-week oral PPS monotherapy could prevent uncomplicated recurrent UTIs up to 24 months after cessation of PPS treatment. We identified a significant improvement in overall QoL in the PPS group. The ongoing phase 3 trial of PPS monotherapy is expected to provide more solid evidence for this promising prophylaxis agent.


In conclusion, nal-IRI/FF is an appropriate treatment option for APC following gemcitabine-containing regimens.Although the combination of nanoliposomal irinotecan plus fluorouracil/folinic acid  exhibited survival benefits in gemcitabine-refractory patients with advanced pancreatic cancer (APC) in the phase III NAPOLI-1 trial, there is limited data on the efficacy and safety of this regimen in real-world settings in Japan. This multicenter, prospective observational study enrolled patients with APC who received nal-IRI/FF after a gemcitabine-based regimen from July 2020 to June 2021. We collected and analyzed clinical data and conducted survival and multivariate analyses. Thirty-one (78%) of the 40 patients had metastases. Nal-IRI/FF was the second-line therapy in 36 patients (90%). The median duration was 3.2 months. The disease control rate was 57%. The median progression-free survival and overall survival (OS) were 4.5 months  and 7.4 months (95% CI: 5.1–10.6), respectively. Common ≥grade 3 toxicities included neutropenia (28%) and fatigue (23%). Fatigue led to treatment discontinuation in 6 out of 10 patients. Multivariate analysis showed that a neutrophil-to-lymphocyte ratio > 4 was a significant risk factor for a short OS (hazard ratio (HR) = 3.08, 95% CI: 1.21–7.85,  According to the 2018 Global Cancer Analysis, pancreatic cancer (PC) remains the fourth leading cause of cancer-related deaths. PC’s 5-year relative survival rate is 8%, which is the lowest among all types of cancer , and values were compared using the Mann–Whitney U test. The Kaplan–Meier curve was drawn, and differences in survival were evaluated using the log-rank test. Univariate and multivariate analyses for survival time were performed using the Cox proportional hazard regression model. Significant variables with a p-value < 0.1 in the univariate analysis were included in the multivariate analysis. All statistical analyses were performed using the Easy R program version1.3.6   months. The median observation period was 7.4 [4.4–0.6] months.The median treatment period was 3.2 [1.7–6.2] months, and the median number of cycles was 6 [3–10]. The administered doses and attributed reasons for dose reduction or discontinuation are summarized in The treatment responses are summarized in p = 0.51) (p = 0.0008) (p = 0.42).Median PFS (mPFS) was 4.5 months (95% CI: 2.8–5.5), and median OS (mOS) was 7.4 months (95% CI: 5.1–10.6) a,b. The  = 0.51) . Regardi 0.0008) a. On thep = 0.08)  tended to have worse survival than patients who continued treatment until disease progression  b.p = 0.08).We compared the clinical characteristics of patients who discontinued nal-IRI/FF due to AEs with those of the patients who did not . The ratp = 0.004), neutrophil-lymphocyte ratio (NLR) > 4 , and body weight loss > 5% from diagnosis  were associated with worse OS in the univariate analysis. The Kaplan–Meier curve (p = 0.02) was an independent predictive factor for shorter survival. There was a significant difference in OS between patients with NLR > 4 and NLR ≤ 4 (p = 0.00003), as shown in We performed univariate and multivariate analyses to identify the possible predictive factors of PFS  and OS . There wer curve c showed In line with previous studies ,12, thisAlthough there was no difference in the CA19-9 response, the PFS and OS observed in the current study were longer than in previous reports. There may be several reasons for this finding. First, the patient population included only a small number of patients with PS = 2 (only one), a high proportion (90%) who received nal-IRI/FF as a second-line therapy, and a relatively high proportion (45%) who received post-study anti-cancer treatment.The subpopulation analysis of NAPOLI-1  reportedSecond, in a previous study on the efficacy of nal-IRI/FF , patientContrary to the NAPOLI-1 study, wherein more than 10% of all patients died during the study, or within 30 days of the last dose, there were no deaths within 30 days of the last dose in the present study. Thus, based on this finding and the fact that ten patients (25%) in our study could continue therapy for more than half a year, the nal-IRI/FF regimen can generally be considered well-tolerated in Japanese clinical practice. Neutropenia and anorexia were common AEs of nal-IRI/FF, as reported in a previous study; there were no significant differences in incidence between this study and the NAPOLI-1 trial . The incIntriguingly, the prognosis in disease-controlled patients with the best response of SD or PR was significantly better than that in patients with progressive disease. The results indicate that the response to the nal-IRI/FF regimen in patients previously treated with a GEM-based regimen considerably impacts prognosis.The survival curve indicated that the discontinuation of nal-IRI/FF due to AEs was possibly associated with shorter OS, although it was not significant in this small population. The PS of patients who discontinued treatment was lower than that of patients who continued the treatment. This finding may help anticipate the clinical benefits of this regimen in individual patients.The present study showed no significant difference in the starting doses of nal-IRI between the patients who continued and discontinued the treatment. However, clinical studies that involved starting dose reductions revealed that these reductions were not significantly associated with PFS or OS . MoreovePS was associated with a short OS, consistent with a meta-analysis reporting that the baseline PS is a potent prognostic factor of PC after progression while on first-line GEM-based regimens . AdditioMeanwhile, other reported factors, such as older age (≥65 years) and the presence of liver metastasis , relatedThis study had several limitations. First, the sample size was too small to reach a definitive conclusion. Second, there was heterogeneity in the treatment dose and schedule due to the observational study design, where modification of the nal-IRI/FF regimen was allowed based on the physician’s decision. Third, we could not compare the efficacy of nal-IRI/FF therapy with that of other regimens such as FFX or modified FFX due to the single-arm fashion of our study. Further comparative studies are required to decide on an optimal regimen for patients with GnP failure. However, our real-world data might help establish an appropriate direction for using nal-IRI/FF.Nal-IRI/FF is an effective sequential treatment after GEM-based treatment in patients with APC. This therapy should be administered under the careful management of AEs, including those triggered by an advanced disease state. The NLR may be a prognostic indicator of the therapeutic effect of nal-IRI/FF.


Lung adenocarcinoma (LUAD), a malignancy with high incidence and mortality rates worldwide, contains multiple genomic and epigenomic abnormalities. And the useful tumor markers associated with these abnormalities need further investigation. Whereas apoptosis is a form of programmed cell death, the expression of apoptosis-related genes in LUAD and its relationship with prognosis is unclear. In the present study, we identified 64 differentially expressed apoptosis-related genes (DEARGs) that were differentially expressed between LUAD tissue and normal lung tissue. Based on these DEARGs, all LUAD cases were classified into two subtypes using The Cancer Genome Atlas (TCGA) cohort to assess the prognostic value of apoptosis-related genes for survival. An 11-gene signature was established by applying the Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression method to construct a multigene prediction model and classify all LUAD patients in the TCGA cohort into high or low AS-score groups. Patients in the low AS-score group had significantly higher survival and prognosis than those in the high AS-score group. Taking the median risk score of the AS-score, LUAD patients in the GSE68465 cohort were divided into two risk groups, low and high. The overall survival (OS) time was longer in the low AS-score group. Combined with clinical characteristics, the AS-score was an independent predictor of LUAD patients. Gene ontology (GO) and Kyoto Encylopedia of Genes and Genomes (KEGG) analyses showed that the differential genes between the two groups were mainly enriched in cellular immunity. Further analysis revealed higher immune checkpoint protein expression and higher tumor mutational burden (TMB) in the high AS-score group, suggesting better efficacy of immunotherapy in the high AS-score group than the low AS-score group. And the high AS-score group was better in chemotherapy and targeted therapy efficiency. In conclusion, the AS-score constructed based on apoptosis-related genes can predict the prognosis of LUAD patients and provide some guidance for the antitumor treatment of LUAD patients. The new Global Cancer Statistics 2020 showed that cancer incidence in China is the highest globally and that lung cancer (LC) remains the second most prevalent malignancy with high mortality . AccordiIt is known that there are two main types of cell death, one is programmed death that is finely regulated by the cell, and the other is uncontrolled cell necrosis . In contThere is increasing evidence that dysregulation of apoptosis signals the development and progression of malignant tumors, which can become resistant to therapeutic agents due to resistance to apoptosis while evading the immune system . The majCurrent evidence suggests that apoptosis not only plays a role in tumorigenesis, cancer metastasis, cancer immunity, and cancer subtypes, but that senescent or lost apoptotic cells are recognized and phagocytosed by macrophages, leading to the release of cytokines that participate in the complex tumor immune microenvironment, which also influences apoptosis .Apoptosis plays an important role in tumor development and antitumor therapy. However, less study of its specific functions and studies in LUAD drug resistance and tumor immune microenvironment are studied. Therefore, we conducted a systematic analysis to explore the prognostic value of apoptosis-related genes in LUAD and investigate the relevance of apoptosis in LUAD to antitumor drugs and the immune microenvironment.BioRender.com) , excluding samples without survival information, including 494 cases of LUAD and 59 normal tissues. Detailed information on these LUAD patients is presented (https://www.genome.jp/kegg/pathway.html), we found 136 apoptosis-related genes according to the apoptosis pathway (map04210). As no complete set of apoptosis-related genes has been reported before, we searched the literature related to apoptosis, performed a comprehensive analysis, and found that all of them could be found in the set of apoptosis-related genes in the kegg database (https://www.ncbi.nlm.nih.gov/geo/) database and was used as the validation set. All data are publicly available online. This study did not require an author to perform experiments on humans or animals. A working diagram showing the overall research process  . The Canresented . In the atabase . GSE6846 process .https://st ring-db. org/) was used to input differential genes for PPI information analysis, and Cytoscape software was used to visualize the PPI network.The “limma” package was used to identify apoptosis-related genes that were differentially expressed between LUAD and normal tissues in the TCGA database. The screening criteria were A false discovery rate (FDR) < 0.05, |logFC| > 0.5 . GO and Consistent clustering identifies apoptosis-related patterns associated with the expression of apoptosis regulators by the k-means method. The number of clusters and their stability is determined by a consensus clustering algorithm using the “ConsensuClusterPlus” package, repeated 1,000 times to ensure classification stability. The prompt function was used to perform principal component analysis. Heat maps and Kaplan-Meyer (KM) curves were plotted using the R packages “heatmap,” “survminer,” and “survival.”The consensus clustering algorithm classifies lung cancer patients into two subtypes, and we next use the R package “limma” to identify differentially expressed genes between subtypes (| logFC | > 2 and FDR <0.001). Univariate Cox regression analysis was used to screen for prognosis-related DEGs, and LASSO—Cox analysis was used to narrow down candidate genes further, resulting in a validated predictive model . AS-scorThe ssGSEA was performed by the “gsva” software package to calculate the infiltration score of 16 immune cells and the activity of 13 immune-related pathways. The data of the Genomics Of Drug Sensitivity in Cancer (GDSC) platform were used to predict the sensitivity of LUAD patients to common chemotherapeutic drugs and targeted therapeutic drugs . The “pRRophetic” R package was used to estimate the half-maximal inhibitory concentration (IC50) .The human LUAD cell lines (A549 and PC9) and the normal human lung epithelial cell line (BEAS-2B) used in this study were provided by the Institute of Cell Research, Chinese Academy of Sciences . The medium for A549 cells was DMEM medium with 10% fetal bovine serum and 1% double antibodies; PC9 and BEAS-2B cells were 1,640 medium with 10% fetal bovine serum and 1% dual antibodies. The cells were placed in a constant temperature incubator with a CO2 concentration of 5% and a temperature of 37°C.Total RNA was extracted by Trizol reagent  according to the instructions. The concentration of the extracted RNA was controlled to be 500 ng/ml with a purity between 1.80 and 2.00. Subsequently, extracted RNA was transcribed using the PrimeScript RT reagent Kit with gDNA Eraser . SYBR Green-based real-time PCR was used for analysis. PCR primers were designed and synthesized by Shanghai Bioengineering Co. (Shanghai.China). Primers used for real-time PCR assays are listed in p < 0.05 was considered statistically significant. Data were processed using R 4.0.5 software.Differences between groups were analyzed using the Wilcoxon test. Independent prognostic analysis was performed using univariate and multivariable cox regression analysis. Correlation tests were performed using Spearman analysis. Survival curves were plotted using log-rank and Kaplan-Meier tests. Mutations between groups were analyzed using the “maftools” R package. The TCGA-LUAD database was downloaded from The Cancer Genome Atlas TCGA public database for a total of 594 samples, and patients with no survival information were excluded from further analysis. Comparing 59 normal tissues with 494 LUAD tissues for DEARGs, a total of 64 differentially expressed genes associated with apoptosis were identified, and the heatmap demonstrated the expression of each differential gene in each sample . Twenty-salmonella infection, and apoptotic pathways and are associated with cysteine-type endopeptidase activity, membrane rafts, and peptide chain endonuclease activity involved in the apoptotic process . The heatmap showed the differential gene expression profile regarding clinical characteristics, including tumor grade, age (≤60 or >60 years), gender, TMN stage, and survival status . The results showed that the C1 group had better clinical performance than the C2 group (p = 0.002). The results suggested that the C1 group has a higher survival rate than the C1 group  . The heC2 group . We alsoC1 group .λ-value and calculated as: AS-score = (−0.003*SERPIND1 exp.) + (−0.118*SFTPC exp.) + (0.033*HMGA2 exp.) + (−0.081*ABCC2 exp.) + (0.178*FBN2 exp.) + (0.117*KRT6A exp.) + (0.022*IL1A exp.) + (−0.030*CYP4B1 exp.) + (0.007*DLGAP5 exp.) + (0.071*C1QL1 exp.) + (0.033*IGF2BP3exp.). The heatmap showed the relationship between 11 model genes and clinical characteristics   .p < 0.001), with the high AS-score group having a lower survival time than the low AS-score group . The high AS-score group had a significantly lower survival time than the low AS-score group , and risk score were all factors associated with prognosis in the TCGA cohort and in the GSE68465 cohort  . MultivaTo further explore differences in gene function and pathways between risk model classifications, we identified differential genes between low AS-scores and high AS-scores in the TCGA cohort. GO enrichment analysis based on these DEGs showed that the DEGs were mainly associated with signaling pathways for the immune response, epidermal development .Increasing studies have shown that tumor metastasis and invasion are inseparable from the tumor microenvironment. Based on the functional analysis, we further explored the role of the constructed AS-score in the immune microenvironment and immunotherapy. Single sample gene set enrichment analysis (ss GSEA) was used to compare the enrichment scores of 16 immune cell types and the activity of 13 immune-related pathways in the TCGA and GSE6846 cohorts in low AS-score versus high AS-score populations. In the TCGA cohort, the high AS-score group had lower levels of immune cell infiltration relative to the low AS-score group, except for p DCs Th1-cells, especially a DCs and I DCs cells. In contrast, in the immune pathway, the high AS-score was mainly enriched in HLA, MHC_class_1, Type_II_IFN_response . SimilarLarge studies have revealed that immunotherapy is emerging as a new hope for cancer treatment, and immune checkpoints play an important role in the immune response . To furtp = 0.0097) (p = 1.9e-05)  of the 238 samples in the high AS-score group had mutations, with the highest mutation frequency being in TTN .In contr 0.0097) . Further1.9e-05) . This stp = 2.2e-09) and paclitaxel (p < 2.22e-16) compared to the low AS-score group (p = 9.2e-08) and gefitinib (p = 0.0013)  , suggestTo better analyze the AS-score signatures of these 11 gene constructs, we predicted their expression in the TCGA cohort. The results showed that CYP4B1 and SFTPC were lowly expressed in LUAD, SERPIND1, HMGA2, ABCC2, KRT6A, IL1A, DLGAP5, C1QL1, IGF2BP3 were highly expressed, while FBN2 was not differentially expressed between LUAD and normal tissues . SubsequAs the most common type of LC, LUAD is currently treated by surgery, radiotherapy, targeted therapy, and immunotherapy .Apoptosis, as a form of programmed cell death (PCD), is mediated through multiple signaling pathways (classified mainly as intrinsic and extrinsic pathways) . The intHowever, it is unclear how apoptosis-associated genes interact in LUAD and whether these genes are associated with clinical characteristics of patients, prediction of antitumor drug efficacy, and infiltration in the immunological microenvironment. Classification of samples based on predefined gene expression profiles is a proven method . In thisIt has been shown that the tumor microenvironment is significantly related to the clinical features, genomic expression, and biological characteristics of tumor patients . A comprCurrent immunotherapy for LUAD is mainly directed at immune checkpoint inhibitors . The mosIn order to further explore the differential impacts on immunity between the high and low AS-score, we compared the TMB. We found that the high AS-score group had more mutational load than the low AS-score group and that AS-score was positively correlated with TMB. The prediction model based on DEARGs showed that the high AS-score group achieved better efficacy in immunotherapy. Similarly, the high AS-score group achieved better efficacy with chemotherapy and targeted drug therapy. At the same time, Ludovic Fournel  found thIn conclusion, our study showed that this AS-score plays a role in clinical prognosis and sensitivity to antitumor drug therapy. Our study provides a new genetic marker for predicting the prognosis of patients with LUAD and provides an important basis for further research into the relationship and new apoptosis-related antitumor drug treatments.